Fig. 3: MCL-1 dependence requires BAX/BAK.
From: Breast cancer dependence on MCL-1 is due to its canonical anti-apoptotic function
A Diagram of experimental set-up for results presented in B. MMTV-PyMT CRISPR/Cas9 Bax/Bak cell line (derivation described in Supplementary Fig. 2A) was injected into mammary fat pad of FVB recipients and tumours allowed to grow to ~5 mm before assignment to vehicle or S63845 treatment groups. S63845 treatment was at 25 mg/kg IV twice per week for 3 weeks. B Mean tumour weight of mice described in A, following 3 weeks of vehicle control (n = 7) or S63845 (n = 7) treatment (25 mg/kg IV twice per week) is shown (±SD), n.s. not significant unpaired t test. C Diagram of experimental set-up for results presented in D–F. MMTV-PyMT; CRISPR/Cas9 Bax/Bak tumour cell line (as used in A, B, this cell line carries RosaCRE-ERT2;Mcl1fl/fl alleles that were not activated in A, B, derivation described in Supplementary Fig. 2A) was injected into mammary fat pad of FVB recipients and tumours allowed to grow to ~5 mm before assignment to vehicle or tamoxifen groups (tamoxifen induces CRE-ERT2-mediated deletion of Mcl1fl/fl); therefore, vehicle treated tumours will be MCL-1 proficient and tamoxifen-induced tumours (TAM) will be MCL-1 deficient. Tumour size was monitored by calliper measurement thrice weekly until harvesting at predefined ethical endpoint (15 mm diameter). D Points indicate individual tumour weights at endpoint, vehicle control (n = 5) or tamoxifen (n = 5). Error bars represent mean ± SD, n.s. not significant unpaired t test. E Kaplan–Meier graph showing tumour-related survival of mice shown in D. Day 0 indicates vehicle/tamoxifen induction (at tumour diameter ~5 mm). Mcl1 deletion in established tumours does not alter survival when Bax/Bak have been CRISPR/Cas9 targeted. Vehicle control (n = 5) or tamoxifen (n = 5). P = 0.2482 Log-rank (Mantel–Cox) test. F Western blot quantification shows sustained decrease in MCL-1 from endpoint tumours shown in D, E. Tumour material sampled for western blot will include host-derived (WT for Mcl1) stroma and donor-derived tumour epithelium. MCL-1 expression is calculated relative to β-actin control for each sample, each point represents an individual tumour and error bars represent mean ± SD. **P < 0.01, unpaired t test. Western blots shown in Supplementary Fig. 3B.
